Mechanisms of thymidine phosphorylase angiogenesis

申请人提供的描述）： 血管生成在实体瘤生长和转移中的作用，以及数据 提示抗血管生成疗法可能是一种可行治疗方法 晚期疾病。胸苷磷酸化酶（TP）最近被发现是 一种血管生成因子，这一观察是基于它与一种 先前描述的血管生成因子、血小板衍生的内皮细胞 生长因子（PD-ECGF）。TP在许多人类实体瘤中以高水平表达， 包括结肠直肠癌在内的肿瘤，其表达水平已经被 在许多临床研究中，包括我们进行的一项研究， 新血管形成、肿瘤侵袭性和患者预后差。 我们和其他人对TP的免疫组织化学评价表明， 在许多但不是全部结肠肿瘤中TP表达升高主要发生在 肿瘤相关巨噬细胞（TAM），而不是肿瘤细胞。我们有 发现人单核细胞系（THP 1）表达较高水平的TP 比人类结肠癌细胞系更好。我们还发现TNF-α刺激了 TP在THP 1细胞和WiDr结肠癌细胞中的表达，我们将使用 这两种细胞系以及TAM的原代培养物，以确定 调节TP表达的分子基础（即转录， 转录后）。TP的血管生成活性似乎依赖于其 催化活性，2-D-脱氧核糖（2dR）作为假定的介质 内皮细胞活化的过程。我们建立了一个体外模型， 人癌细胞或单核细胞可以与正常人共培养 内皮细胞（HUVEC），并可以诱导其迁移的TP依赖性 方式我们将使用这个模型来开始确定TP 刺激HUVEC和人微血管内皮细胞（HMEC）迁移， 使用胸苷和2dR的类似物，我们将合成以检验假设 迁移仅与2dR形成有关，通过使用TP抑制剂， 我们已经制造了中和TP抗体来测试TP 细胞内作用足以刺激HUVEC迁移。小 进一步了解TP的作用机制。我们的研究显示 介导TP与VEGF诱导的HUVEC的特异性整合素的差异 迁移是首次探索TP对内皮细胞的细胞作用， 细胞我们还发现TP诱导FAK的酪氨酸磷酸化 粘着斑激酶（focal adhesion kinase）是一种在细胞凋亡中起核心作用的蛋白质， 附着、迁移和信号传导。我们会继续研究， TP激活的整合素相关信号转导通路 在HUVEC和HMEC中，测试这些最初不同于 那些发生在对血管生成因子如VEGF和bFGF的反应中， 都有明确的细胞表面受体除了提供基本的 了解TP结肠癌血管生成作用的机制，这些 研究可以提供见解，以优化使用当前和未来的 靶向整联蛋白的抗血管生成剂。

DESCRIPTION provided by applicant): There is an increasing appreciation of the role of angiogenesis in the growth and metastasis of solid tumors, and data suggest that antiangiogenesis therapy could be a viable approach to treatment of advanced disease. Thymidine phosphorylase (TP) has been recently found to be an angiogenic factor, an observation that was based on its identity to a previously described angiogenic factor, platelet-derived endothelial cell growth factor (PD-ECGF). TP is expressed at high levels in many human solid tumors including colorectal cancers, and the level of its expression has been correlated in numerous clinical studies, including one we conducted, with neovascularization, tumor aggressiveness, and poor patient prognosis. Immunohistochemical evaluation of TP by us and others has demonstrated that elevated TP expression in many, but not all, colon tumors occurs mainly in tumor-associated macrophages (TAMs), rather than in the tumor cells. We have found that a human monocytic cell line (THP1) expressed a higher level of TP than human colon carcinoma cell lines. We also found that TNF-alpha stimulated TP expression in THP1 cells and in WiDr colon carcinoma cells, and we will use these two cell lines as well as primary cultures of TAMs to determine the molecular basis for the regulation of TP expression (i.e. transcriptional, post-transcriptional). TP's angiogenic activity appears to be dependent on its catalytic activity, with 2-D-deoxyribose (2dR) serving as the putative mediator of endothelial cell activation. We have established an in vitro model in which human cancer cells or monocytes can be co-cultured with normal human endothelial cells (HUVEC), and can induce their migration in a TP-dependent manner. We will use this model to begin to determine the mechanisms by which TP stimulates HUVEC and human microvascular endothelial cell (HMEC) migration, using analogs of thymidine and 2dR we will synthesize to test the hypothesis that migration is solely related to 2dR formation, and by using a TP inhibitor we have made and a neutralizing TP antibody to test the hypothesis that TP's intracellular actions are sufficient to stimulate HUVEC migration. Little further is known of the mechanism of action of TP. Our studies showing differences in the specific integrins which mediate TP vs. VEGF-induced HUVEC migration are the first to explore the cellular actions of TP on endothelial cells. We have also found that TP induces tyrosine phosphorylation of FAK (focal adhesion kinase), a protein that plays a central role in cell attachment, migration, and signaling. We will continue our studies to define the integrin-associated signal transduction pathways that are activated by TP in HUVEC and HMEC, testing the hypothesis that these differ initially from those occurring in response to angiogenic factors such as VEGF and bFGF, which have clearly defined cell-surface receptors. In addition to providing a basic understanding of the mechanisms of angiogenic action of TP colon cancers, these studies could provide insights to optimize the use of current and future anti-angiogenic agents which target integrins.